Extended haplotypes in the complement factor H (CFH) and CFH-related (CFHR) family of genes protect against age-related macular degeneration: characterization, ethnic distribution and evolutionary implications

Abstract

Background: Variants in the complement factor H gene (CFH) are associated with age-related macular degeneration (AMD). CFH and five CFH-related genes (CFHR1-5) lie within the regulators of complement activation (RCA) locus on chromosome 1q32. Aims and Methods. In this study, the structural and evolutionary relationships between these genes and AMD was refined using a combined genetic, molecular and immunohistochemical approach.

Results: We identify and characterize a large, common deletion that encompasses both the CFHR1 and CFHR3 genes. CFHR1, an abundant serum protein, is absent in subjects homozygous for the deletion. Genotyping analyses of AMD cases and controls from two cohorts demonstrates that deletion homozygotes comprise 1.1% of cases and 5.7% of the controls (chi-square=32.8; P= 1.6 E-09). CFHR1 and CFHR3 transcripts are abundant in liver, but undetectable in the ocular retinal pigmented epithelium/choroid complex. AMD-associated CFH/CFHR1/CFHR3 haplotypes are widespread in human populations.

Conclusion: The absence of CFHR1 and/or CFHR3 may account for the protective effects conferred by some CFH haplotypes. Moreover, the high frequencies of the 402H allele and the delCFHR1/CFHR3 alleles in African populations suggest an ancient origin for these alleles. The considerable diversity accumulated at this locus may be due to selection, which is consistent with an important role for the CFHR genes in innate immunity.

Figure 1

The CFH gene contains 20 short consensus repeats (SCR), or complement control modules, each of which encodes a functional domain of ~60 amino acids. In addition to the full-length transcript, there is an alternatively-spliced, truncated gene product of CFH that has sequence identity with SCRs 1–7. An 8^th exon spliced to SCR 7 in the truncated variant (black rectangle) encodes a stop codon, a unique untranslated region, and 4 C-terminal amino acids (SFTL). The full length and truncated forms of CFH transcripts are referred to as variants 1 and 2, and the respective encoded proteins are designated as isoforms a and b. The 20 SCRs of CFH encode a number of functional domains. The complement Factor I co-factor and decay accelerating activities reside within SCRs 1–4 (boxed). Three C3b binding sites are associated with SCRs 1–4, 12–14, and 19–20 (shown in yellow). A RGD sequence in SCR 4 mediates cell adhesion through binding to complement receptor 3 (CR3), an αMβ2 integrin. A binding site for C-reactive protein (CRP), has been localized to the region spanning SCRs 7–11 (blue; in brackets), and a binding site for the Streptococcus pneumoniae surface protein β lies within a domain encompassing SCRs 8–11 (not shown). Low and high affinity adrenomedullin (AM) binding sites have been mapped to SCRs 8–11 and SCRs 15–20 respectively. Three heparin binding sites have been localized to SCRs 7, 12–14, and 20. Also located within SCR 20 is a sialic acid binding site (not shown). The 5 related members of the CFH gene family are represented by 5 separate genes (gene symbols CFHR1-5) all of which reside within the regulators of complement activation (RCA) gene cluster on chromosome 1q32. Each of the 5 CFH-related genes contains a subset of the 20 CFH SCR domains (see Zipfel, Skerka et al, 2002 for review). The amino acid sequence homologies of CFHR1-5 SCRs to their CFH counterparts are shown as a percentage in white numerals above each SCR. With the exception of CFHR4 where SCR 7 is absent, all members of the CFH family contain two conserved regions corresponding to SCRs 6–7 and 19–20 of CFH (highlighted in red), and no members contain domains corresponding to SCRs 1–4 of CFH. Based upon their close structural relationships, it may be predicted that the five CFH -related family members also share functional similarities with CFH.

Figure 2

A representative Western blot of serum proteins from 10 of 144 individuals analyzed by nonreducing SDS-PAGE and chemiluminescent immunoblotting. (Panel A) CFH is visualized as the faster migrating of the two bands (~150 kDa; identified as FH by co-migration with purified CFH; not shown) of two immunoreactive bands in the high molecular weight region of the gel/blot. The nature of the higher molecular weight CFH antibody-reactive band was not determined. The blot depicted was exposed to film for 3 seconds. (Panel B) CFHR1a, one of the most abundant proteins in serum, as well CFHR2 and the truncated form of CFH (CFHT/FHL-1) can be distinguished from CFH following separation of proteins in the lower molecular weight region of the gel/blot. The faster migrating band is CFHR-1β. No immunoreactive CFHR1 is present in the serum of three patients (lanes 2, 6 and 10), two unaffected patients possessing 1277TT genotypes (protective CFH H4 haplotype) and one patient with a 1277CT genotype, respectively. Subsequent SSCP analysis and direct DNA sequencing showed that both individuals both have a homozygous deletion of the CFHR1 and CFHR3 genes. The blot was exposed to film for 7 minutes.

Figure 3

A haplotype network resulting from the analysis of variants in the CFH locus is shown. The size of the sphere is proportional to the estimated frequency of the haplotype. Haplotypes adjacent to each other are predicted to arise from a single mutational step. The small spheres represent nodes that were not observed, but are predicted. The 402H haplotype is depicted, along with the two major protective haplotypes, and the haplotypes that are neutral for AMD risk.

Figure 4

A schematic map of that portion of the RCA cluster on 1q31 showing the relative positions of the CFH, CFHR1 and CFHR3 genes. The solid green boxes represent a segmental duplication of 28 kb that occurred within this locus. The primers employed to define the CFHR1/CFHR3 deletion boundaries are shown in Supplemental Table 1; “+” indicates products that amplify in deletion homozygotes and “−” regions that fail to amplify in deletion homozygotes.